Opioids are the most effective treatment for pain. Unfortunately, their efficacy decreases with repeated administration because of the development of tolerance. The broad long-term objective of this research is to understand the underlying neural mechanisms for tolerance so more effective pain treatments can be developed. Many previous studies have examined opioid tolerance. However, these studies show over 30 different mechanisms for tolerance, and it is unlikely that all of these putative mechanisms are directly involved. The proposed studies will focus on tolerance in the ventrolateral periaqueductal gray (PAG) in an attempt to identify causal mechanisms for tolerance. PAG neurons are particularly well suited for studying opioid tolerance because microinjection of morphine into the PAG produces antinociception and repeated microinjections produce tolerance. In addition, the neuronal circuitry and intracellular signaling cascades through which opioids produce antinociception have been described. The proposed studies will take advantage of both intracellular and behavioral measures of tolerance in an attempt to understand the neurochemical basis of tolerance in these neurons. Tolerance could be caused by a change anywhere along the signaling pathway from opioid receptors to feedback circuits. The aims of this project will examine all of the steps along this pathway. Aim 1 will identify the specific PAG neurons that contribute to opioid tolerance. Aim 2 will determine whether receptor internalization contributes to the development of tolerance. Aim 3 will identify the intracellular molecules that contribute to the development of tolerance. And Aim 4 will determine whether feedback to the PAG is necessary for the development of tolerance. A unique feature of this project is that whole cell recordings will be used to link neuronal changes to behavioral measures of tolerance. For example, the intracellular signaling cascades believed to contribute to tolerance will be systematically disrupted to determine the contribution of each step in this signaling cascade to opioid tolerance measured behaviorally. The combined use of in vivo and in vitro techniques provides a powerful approach to understanding the link between molecular changes and behavioral consequences. In the long term, such knowledge will contribute to the development of therapies that improve pain treatment by preventing tolerance to opioids.